Fig. 3. Cell morphology and expression of adipogenic-associated markers in non-myogenic (left column) and myogenic (right column) clones. (A,B) Fluorescent images of 7-day-old clones derived from one GFP-myofiber; the clone depicted in panel A shows multiple cell morphologies and the clone depicted in panel B shows myotubes. (C,D) Representative merged images of 2-week-old cultures visualized with DAPI (blue) and oil-red-O (red); adipogenic cells are detected only in the non-myogenic clone. (E,F) Images of 2-week-old cultures labeled with anti-PPAR (red) and DAPI (blue) merged with the phase-contrast image; intense nuclear staining of PPAR was evident only in the adipogenic cells (E); a very low level of PPAR was detected in some of the myonuclei (F). (G-H') Double immunostaining with c/EBP (green) and MyoD (red), image of each stain was merged with the phase-contrast image; the non-myogenic clone was additionally stained with oil-red-O; both clones exhibit similar levels of nuclear expression of c/EBP (G and H) but only the myogenic clone expresses MyoD (G',H'). Note that the two mononucleated cells in panel H' that seem to be negative, in fact express low levels of MyoD. However, this weaker staining is lost when the MyoD immunostaining image and the phase-contrast image are merged. The variations in the level of MyoD probably reflect different phases of the cell cycle as previously reported (Kitzmann et al., 1998). Of particular interest is the pair of cells (top of micrograph in H') that seem to be just separating from each other following cell division with only one cell expressing a high level of MyoD. Bar, 20 µm.